Project Summary Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal disease, efficient therapies for which are mostly limited to lung transplantation. It is unclear why even in individuals with genetic risk factors, clinically important lung disease seldom manifests until the later decades of life and why many high risk individuals are spared. Recently, several groups of investigators have shown that tissue-resident alveolar macrophages originate from fetal monocytes and populate alveolar space soon after birth and maintain their population throughout the lifespan via self-renewal, without contribution from circulating adult bone marrow- derived monocytes. These ?tissue-resident alveolar macrophages reciprocally interact with cells in the alveolar epithelium to dampen the immune response to endogenous and environmental challenges in the lung, which can threaten gas exchange. However, in response to injury, when tissue-resident alveolar macrophages are depleted, bone marrow-derived monocytes are recruited to the lung where they differentiate into ?monocyte- derived? alveolar macrophages and orchestrate a pro-inflammatory response. Using unique tools we developed to unambiguously label tissue-resident and monocyte-derived alveolar macrophages in the lungs of mice, we show that monocytes are recruited to the lung during injury where they differentiate into monocyte- derived alveolar macrophages and persist for months after the original injury. To explore the role of monocyte- derived macrophages in humans with lung fibrosis, we have developed a systematic protocol to isolate macrophage populations and alveolar type II cells from the samples of the human lung obtained at the time of lung transplant for transcriptomic analysis and adoptive transfer studies into humanized mouse model. We will use these tools to test the hypothesis that the loss of tissue-resident alveolar macrophages and their replacement by monocyte derived-alveolar macrophages contributes to the enhanced susceptibility to fibrosis. Aim 1: To determine whether TR-AM or Mo-AM differentially contribute to the development of lung fibrosis. Aim 1.1: To test if cell ontogeny determines the response of alveolar macrophages to a subsequent profibrotic stimulus. Aim 1.2: To determine if the lung microenvironment at the time of lung repopulation programs Mo-AM to respond differently to a subsequent fibrotic challenge. Aim 2: To determine whether the contribution of alveolar macrophages to lung fibrosis is cell autonomous or is shaped by the local microenvironment of the fibrotic lung. Aim 2.1: To determine if TR-AM will retain their anti- inflammatory/non-fibrotic phenotype in a fibrotic lung microenvironment. Aim 2.2: To determine whether monocytes and/or alveolar macrophages from healthy donors and patients with IPF demonstrate cell- autonomous differences in gene expression and whether these persist over time.